Invasive amebiasis, a disease caused by the enteric protozoan Entamoeba histolytica is a major public health problem in developing countries (32). Recently, progress has been made in understanding the molecular bases of the pathogenesis of this disease. Thus, it has been shown that attachment of E. histolytica trophozoites to mammalian cells, mediated by a surface lectin, is required for direct-contact amebic cytolytic activity (20, 21, 22). An amebic protein which forms ion channels in the membranes of target cells and probably participates in cytotoxicity has also been identified (15, 33). In addition, proteolytic enzymes present on the parasite surface have been implicated in the disruption of the intestinal extracellular matrix (8, 14, 18, 19, 24).
Since collagen is a major component of the extracellular matrix, in previous works we focused our attention on the E. histolytica collagenase (16, 18, 19, 24). The levels of this enzyme correlate with the virulence of different E. histolytica strains (8, 19). The collagenase is specific for type I collagen and is localized in the plasma membrane of the trophozoite. In addition, the collagenolytic activity in trophozoites increases when they are cultured in the presence of collagen (16). In recent in vitro studies we have shown that the increase in collagenolytic activity is accompanied by the intracellular formation of electron-dense granules (EDGs). These granules accumulate in the parasite plasma membrane and are subsequently released into the extracellular milieu (16),
Differences between pathogenic and non-pathogenic E. histolytica isolates have been observed recently using monoclonal antibodies (26) and DNA probes (3, 9, 27). However, until now there are not practical techniques to manage clinical patients.
The applicant has observed, that EDGs secreted by the parasite E. histolytica, contains an unknown collagenolytic activity specific for collagen type I. Consequently, this activity has been correlated with human tissue invasion.
In addition, this activity correlates with the trophozoite virulence, therefore it was considered that one or several of the EDG components may be useful in the diagnosis of pathogenic E. histolytica trophozoites. To test this possibility, EDGs were isolated by differential centrifugation and used as immunogen to prepare polyclonal and monoclonal antibodies specific for EDG antigens (LMT). Polyclonal antibodies were prepared from the serum of a goat hyperimmunized with EDGs. Monoclonal antibodies were prepared by fusion of Balb/c mice (immunized with EDG protein) spleen cells and the cell line SP2/O-Ag14. These antibodies were specific for EDG by ELISA, immunofluorescence and immunoelectron microscopy. These antibodies recognized E. histolytica from strains HM1, HM38, HK9, but did not react with the non-pathogenic trophozoites E. moshkovskii, E. invadens, Laredo and E. histolytica from asymptomatic patients in xenic culture.